Determining SARS-CoV host gene requirements by mutagenesis and RNA interface

通过诱变和 RNA 界面确定 SARS-CoV 宿主基因要求

• 批准号: 7274613
• 负责人: ROBERT J HOGAN
• 金额: $ 23.61万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7274613
• 关键词: Avian Influenza; Award; Biology; Candidate Disease Gene; Cell Line; Cell Survival; Cells; Cowpox; Data; Developing Countries; Development; Disease; Disruption; Economics; Essential Genes; Gene Expression; Gene Silencing; Generations; Genes; Genetic; Goals; Growth; HIV-1; Incidence; Infection; Influenza; Insertional Mutagenesis; Intervention; Kinetics; Libraries; Life Cycle Stages; Long-Term Effects; Measles; Mediating; Messenger RNA; Morbidity - disease rate; Mutagenesis; Mutate; Mutation; Pathogenesis; Pathway interactions; Phenotype; Plaque Assay; Polymerase Chain Reaction; Positioning Attribute; Proteins; Public Health; RNA; RNA Interference; Rate; Reovirus; Research; Resistance; Role; SARS coronavirus; Screening procedure; Small Interfering RNA; Source; Survivors; Technology; Therapeutic; Therapeutic Intervention; Time; Viral; Virus; Virus Diseases; Work; cell growth; cellular targeting; cytotoxic; design; innovation; killings; knock-down; mortality; novel; novel vaccines; pathogen; prevent; prophylactic; prospective; respiratory infection virus; small hairpin RNA; tool

DESCRIPTION (provided by applicant): Respiratory virus infections are a major source of morbidity, mortality, and economic loss in both developed and under-developed countries. The emergence of SARS-CoV and the increased incidence of avian influenza highlight the potential importance of new vaccines and therapeutics to prevent disease and disease spread around the world. The rationale for this study is that SARS-CoV, like all other obligate intracellular pathogens, requires host proteins for its life cycle and that inhibiting the expression of critical host proteins should block viral replication. We have successfully used gene trap insertional mutagenesis to randomly inactivate cellular genes as a tool for rapidly identifying novel host genes required for influenza A, HIV-1, Marburg, Ebola, or reovirus replication, but not host cell survival. Viral infection normally kills the chosen host cell line unless a gene essential for the viral life cycle has been inactivated. We identified several pathways used by unrelated viruses suggesting the existence of conserved mechanisms for viral replication, and that inhibiting such pathways using small interfering RNA (siRNA) may allow broad spectrum therapeutic intervention. The objectives of the current application are to extend gene trap technology towards the study of novel host genetic requirements for SARS-CoV infection, and to use RNA interference as a strategy to independently confirm the requirement of these genes in viral replication. Our Specific Aims are to 1) use Vero E6 cell gene trap libraries to select cells resistant to SARS- CoV infection and identify candidate genes whose disruption confer the resistant phenotype, and 2) to characterize the effects of silencing candidate genes identified in Specific Aim 1 on SARS-CoV replication and host cell viability and growth rates. This study is designed to identify specific siRNAs that inhibit SARS-CoV replication without causing detrimental effects to host cells. The benefit to public health is that the transient knock- down of host gene mRNAs to block infectious pathogens could confer broad spectrum therapeutic intervention that is difficult, if not impossible, to circumvent through mutation of the pathogen. The goal of this application is to determine novel host genetic requirements essential for SARS-CoV replication. Candidate host genes performing critical roles in SARS-CoV replication will be identified by randomly mutating host genes, and then identifying disrupted genes in surviving clonal SARS-CoV-resistant cell lines. The requirement of candidate genes in SARS-CoV replication will be confirmed in RNA interference (RNAi) studies. Following RNAi screening, cell lines expressing short hairpin (shRNA) against critical genes will be generated to determine the effects of long term target gene silencing on cellular growth rates and viability, and to prepare for prospective mechanistic studies beyond the term of this application.

描述（由申请人提供）：呼吸道病毒感染是发达国家和不发达国家发病、死亡和经济损失的主要来源。 SARS-CoV 的出现和禽流感发病率的增加凸显了新疫苗和治疗方法对于预防疾病和疾病在世界范围内传播的潜在重要性。这项研究的基本原理是，SARS-CoV 与所有其他专性细胞内病原体一样，在其生命周期中需要宿主蛋白，并且抑制关键宿主蛋白的表达应该阻止病毒复制。我们已经成功地使用基因陷阱插入诱变来随机灭活细胞基因，作为快速识别甲型流感、HIV-1、马尔堡病毒、埃博拉病毒或呼肠孤病毒复制所需的新宿主基因的工具，但不能识别宿主细胞存活所需的新宿主基因。病毒感染通常会杀死所选的宿主细胞系，除非病毒生命周期必需的基因已失活。我们确定了不相关病毒使用的几种途径，表明存在病毒复制的保守机制，并且使用小干扰RNA（siRNA）抑制这些途径可能允许广谱治疗干预。当前应用的目标是将基因捕获技术扩展到SARS-CoV感染的新宿主遗传要求的研究，并使用RNA干扰作为独立确认这些基因在病毒复制中的要求的策略。我们的具体目标是 1) 使用 Vero E6 细胞基因捕获库来选择对 SARS-CoV 感染具有抵抗力的细胞，并识别其破坏赋予抵抗表型的候选基因，以及 2) 表征特定目标 1 中确定的沉默候选基因的效果SARS-CoV 复制以及宿主细胞活力和生长率。本研究旨在鉴定抑制 SARS-CoV 复制而不会对宿主细胞造成有害影响的特定 siRNA。对公共健康的好处是，通过短暂敲低宿主基因 mRNA 来阻断传染性病原体，可以提供广谱治疗干预，而通过病原体突变很难（如果不是不可能）规避这种治疗干预。该应用的目标是确定 SARS-CoV 复制所必需的新宿主遗传要求。将通过随机突变宿主基因来鉴定在 SARS-CoV 复制中发挥关键作用的候选宿主基因，然后识别存活的克隆 SARS-CoV 抗性细胞系中被破坏的基因。 SARS-CoV 复制中候选基因的需求将在 RNA 干扰 (RNAi) 研究中得到证实。 RNAi筛选后，将产生表达针对关键基因的短发夹（shRNA）的细胞系，以确定长期靶基因沉默对细胞生长速率和活力的影响，并为本申请期限之外的前瞻性机制研究做好准备。